The long-term goal of this research is to determine how environmental signals such as temperature regulate morphology and virulence in the fungal pathogen Histoplasma capsulatum. H. capsulatum grows in a filamentous form in the soil; once inhaled into a mammalian host, these cells switch their growth program to a parasitic yeast form that subverts the innate immune system to cause disease. A similar switch from soil to host form is observed for the evolutionarily related systemic dimorphic fungal pathogens, which include H. capsulatum as well as select agent pathogens such as Coccidioides species. For all of these pathogens, temperature is a key signal that regulates this morphogenetic switch, which is thought to be essential for H. capsulatum virulence. By elucidating how H. capsulatum cells sense and respond to host temperature, we will define critical molecular landmarks that promote changes in morphology as well as the expression of virulence traits. These studies will shed light on fundamental processes such as signal transduction and gene regulation, as well as uncover the role of temperature-dependent pathways in fungal pathogenesis. Over the last funding period, we identified the first transcriptional regulators required for growth in the yeast form in response to host temperature. These factors, named Ryp1, Ryp2, and Ryp3, are homologous to key developmental regulators in other fungi, and represent critical elements of the temperature-dependent regulatory circuit in H. capsulatum. Interestingly, the precise biochemical function of the orthologous regulators in other fungi is unclear, thus adding additional significance to our functional characterization in H. capsulatum. We have recently shown that the Ryp proteins associate with the upstream regions of both morphology and virulence genes, suggesting that they directly regulate essential components of the morphology and virulence programs. Furthermore, these data provide the first molecular evidence that the transcriptional regulation of morphology and virulence factors is coupled. In this proposal, our specific aims are to investigate and identify (1) the molecular mechanism of activation of the Ryp proteins by temperature; (2) the mechanisms by which the Ryp proteins regulate gene expression; and (3) the elements of the Ryp-dependent regulatory circuit that lie downstream of these factors and their effect on morphology and virulence. Taken together, the resultant data will greatly enhance our understanding of the molecular response pathway of H. capsulatum to host temperature. Additionally, we will gain valuable knowledge about individual morphology and virulence factors that are required for H. capsulatum pathogenesis in the host.